The synthesis of polypeptides by conventional solid-phase methods, amino acid by amino acid, is limited by low yields when the polypeptides synthesized are large. In order to overcome this limitation, it is known to assemble two polypeptides by chemical ligation in order to produce a longer polypeptide.
Total polypeptide synthesis is increasingly useful for preparing proteins with well-defined structures or bearing natural modifications, such as post-translational modifications, or unnatural modifications. Chemical ligation methods respond to this need; however they prove limited in their use and their industrial application.
Generally, in these methods, it is desirable for the link between the polypeptides assembled by ligation to be native, i.e. to correspond to the natural structure of the polypeptides.
The main native ligation method currently in existence is that of Kent and Dawson, described for example in international applications WO 96/34878 and WO 98/28434. This method is based on a chemoselective reaction between a (C-terminal) peptide thioester and a cysteinyl-peptide. Nevertheless, the main drawback of this method is the production of the peptide thioesters which require complex chemical processes. These methods of the prior art are unsatisfactory because they inevitably lead to mixtures that may be difficult to separate, thus affecting the purity of the end product obtained, and to inevitable losses in yield.
An alternative method is the ligation known as the Staudinger reaction, described in international applications WO 01/68565 and WO 01/87920. This comprises the reaction of a phosphinothioester with an azide and the hydrolysis of the combined reagents in order to form an amide bond. However this method is difficult to apply on an industrial scale.
Another method, described in international application WO 2007/037812, is based on the reaction of an α-ketoacid with an N-alkoxyamine in a decarboxylative condensation reaction. However, the ketoacids are molecules which are difficult to produce and incorporate into peptides. Also, this third method is difficult to apply in peptide synthesis laboratories that do not have the means to carry out complex organic syntheses.
The document WO 2011/051906 describes a native ligation process involving a thiol compound. Although this method is highly satisfactory, the reaction kinetics of this thiol compound are sometimes slow, in particular when the amino acids surrounding the bond between the two peptides are hindered.
A need therefore remained for a native ligation method provided with rapid reaction kinetics so as to facilitate the ligation of peptides by the formation of a bond between two hindered amino acids.